RNA_misc_01

30!

10!

Specific sequences (cis-elements) in the nascent RNA serve as splice sites

• The first 2 bases of introns are GU (= GT in the DNA)
• The last 2 bases are AG
• The branch site in the middle of intron is an invariant A
• 

• 2’-OH group of adenosine nucleotide in the middle of intron attacks phosphodiester bond between 1st exon & G at the beginning of intron
• Forms loop of the lariat
• Separates first exon from intron
• 

• 3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exon
• Forms the exon-exon phosphodiester bond
• Releases intron in lariat form at the same time
• 

The conserved branch-point sequence is essential for splicing and it designates the closest downstream AG as the 3’ splice site

the lariat is formed

exons are ligated

• True
• knockdowns of the minor spliceosome are lethal!

Survival of Motor Nuerons (SMN) protein complex!

because the splicing of minor introns is inefficient (and thus represents a rate-limiting step in the processing of the pre-mRNAs that harbor them)

• Ribosomal RNA
• structural and functional components of ribosomes

small nucleoular RNA

• 5S+5.8S+28S+proteins
• 

• 18S+proteins
• 

Helix 69 is a hairpin structure in the large subunit of the ribosome that interacts with the helix 44 of the small ribosomal subunit and with tRNA

• helix 44 is a hairpin structure in the small ribosomal subunit and interacts with tRNA
• also interacts with Helix 69 in the large subunit of the ribosome

• pseudouridilation
• 2'-O-methylation
• m6A
• m7G
• m1A
• m5C

• base-pair with their target rRNA (as well as with tRNA or snRNA) and assist in cleavage and/or modification
• Some snoRNAs are expressed from their own promoters by RNA polymerase II or III
• The large majority of snoRNAs are encoded by the introns of protein-coding genes involved in ribosome synthesis or translation
• Some snoRNAs are introns spliced from apparently nonfunctional mRNAs (the genes encoding these mRNAs seem to exist only to express snoRNAs from excised introns!)

transfer RNA

• RNAP III
• tRNAs are transcribed by RNA polymerase III as pre-tRNAs and are extensively processed in the nucleoplasm

• tRNAs are transcribed by RNA polymerase III as pre-tRNAs and are extensively processed in the nucleoplasm
• The 5’-end sequence of pre-tRNA  is removed by RNase P (a ribonucleoprotein containing a catalytic RNA)
• The 3' end is removed by the RNase Z enzyme
• Non-template CCA is added to the 3’- end by nucleotidyl transferase
• Multiple internal bases are modified by enzymes and snoRNPs (uridine -> dihydrouridine, pseudouridine, or ribothymidine and many others)
• Some pre-tRNAs contain a short intron within the anticodon loop: it is removed by tRNA splicing endonuclease, i.e. via a mechanism distinct from the splicing of pre-mRNA and self-splicing introns

• A 14-nucleotide intron in the anticodon loop is removed by a tRNA splicing endonuclease
• A 16-nucleotide sequence at the 5’-end is cleaved by RNase P
• U residues at the 3’-end are removed by RNase Z  and replaced by the CCA sequence with the help of nucleotidyl transferase
• Numerous bases in the stem-loops are converted to characteristic modified bases (e.g., D = dihydrouridine, Ψ = pseudouridine) by specific enzymes (e.g., pseudouridine synthase) with the help of snoRNAs

• Pre-tRNA is cleaved by a tRNA splicing endonuclease at each side of the intron, thereby excising the intron and generating a 2’,3’-cyclic phosphomonoester at the 3’-end of the 5’ exon
• The multistep reaction of joining the two exons requires two nucleoside triphosphates: a GTP and an ATP
• GTP (hydrolyzed by kinase) contributes the phosphate group for the 3’ → 5’ linkage in the finished tRNA molecule
• ATP is required for the activation of the tRNA ligase that joins the two exons
• The 2’-phosphate on the 5’ exon is removed by the 2-phosphotransferase

• Occur during and/or after transcription (co- and post-transcriptionally)
• Involve a large number of proteins and non-coding RNAs (often in a form of ribonucleoprotein particles, or RNPs)

post-transcriptional alteration of the sequence of nucleotides in the RNA

• RNA modification, i.e., enzymatic alteration of individual nucleotides (common!)
• insertion/deletion of nucleotides in the RNA (rare!)
• Some RNA modifications (C-to-U and A-to-I) alter RNA coding capacity -> aka substitution or base editing

RNA modifications

genome level studies of RNA modifications

• Immunoprecipitation with modification-specific antibodies (RIP-seq)
• mass spec (but not genome level detection)
• Chemical  treatments (e.g., with click chemistry) that block reverse transcription (Chem-seq)
• Next-gen sequencing: Illumina to locate RT errors in cDNA or Oxford Nanopore to detect electric signal deviations in RNA squiggles

• Chemical derivatization is used: purified RNA is treated with carbodiimide CMCT (B) that reacts with pseudouridine (A) and forms a bulky adduct (C)
• The adduct can be detected by mass-spec or by reverse transcription and sequencing (D)
• 

• In males, splicing factor SPF45 binds to AG dinucleotide in the 3’ splice site and promotes inclusion of the 3rd exon (with a premature stop codon) => 2,3,4
• In females, SXL binds to a polypyrimidine tract, interacts with the AG-bound SPF45 and inactivates it, leading to exclusion of the 3rd exon => 2,4
• 

• In males, splicing factor U2AF binds to a polypyrimidine tract in the 3’ end of intron and promotes splicing at the proximal site of the 2nd exon => male Tra mRNA contains an early stop codon
• In females, SXL (a splicing repressor) binds to the polypyrimidine tract, thus blocking access of U2AF to this splice site; U2AF binds and promotes splicing at an alternative distal 3’ splice site => female functional Tra protein
•